Starch comprises amylopectin and amylose. Amylopectin is a highly branched carbohydrate polymer with short α-(1→4)-D-glucan chains which are joined together at branch points through α-(1→6) linkages forming a branched and bushlike structure. On average, there is one branch point for every 20-25 α-(1→4) linked glucose residues. In contrast, amylose is a linear structure mainly consisting of unbranched α-(1→4)-D-glucan units. Typically, starches contain about 75% amylopectin molecules and about 25% amylose molecules.
More specifically, linear malto-oligosaccharides are composed of 2-10 units of α-D-glucopyranose linked by an α-(1→4) bond. Due to their properties such as low sweetness, high waterholding capacity, and prevention of sucrose crystallisation [1] these compounds have potential applications in the food industry. The preparation of malto-oligosaccharides with a degree of polymerisation (DP) above 3 (i.e. DP>3) in larger amounts is however tedious and expensive.
As background information, DP1=glucose, DP2=maltose, DP3=maltotriose, DP4=maltotetraose, DP5=maltopentaose, DP6=maltohexaose, DP7=maltoheptaose, DP8=maltooctaose, DP9=maltononaose, and DP10=maltodecaose.
The discovery of microbial enzymes, which produce malto-oligosaccharides of a specific length could allow the production of larger amounts of these oligosaccharides [2].
Amylases are starch-degrading enzymes, classified as hydrolases, which cleave α-D-(1→4) O-glycosidic linkages in starch. Generally, α-amylases (E.C. 3.2.1.1, α-D-(1→4)-glucan glucanohydrolase) are defined as endo-acting enzymes cleaving α-D-(1→4) O-glycosidic linkages within the starch molecule in a random fashion [3]. In contrast, the exo-acting amylolytic enzymes, such as β-amylases (E.C. 3.2.1.2, α-D-(1→4)-glucan maltohydrolase), and some product-specific amylases cleave the starch molecule from the non-reducing end of the substrate [4]. β-Amylases, α-glucosidases (E.C. 3.2.1.20, α-D-glucoside glucohydrolase), glucoamylase (E.C. 3.2.1.3, α-D-(1→4)-glucan glucohydrolase), and product-specific amylases can produce malto-oligosaccharides of a specific length from starch.
Several amylases producing malto-oligosaccharides of a specific DP have been identified previously including maltohexaose-producing amylases from Klebsiella pneumonia [5, 6], Bacillus subtilis [7], B. circulans G-6 [8], B. circulans F-2 [9, 10], and B. caldovelox [11, 12]. Maltopentaose-producing amylases have been detected in B. licheniformis 584 [13] and Pseudomonas spp. [14, 15]. Furthermore, maltotetraose-producing amylases have been reported from Pseudomonas stutzeri NRRL B-3389 [16, 17], Bacillus sp. MG-4 [18] and Pseudomonas sp. IMD353 [19] and maltotriose-producing amylases from Streptomyces griseus NA-468 [20] and B. subtilis [21].
EP-B1-298,645 describes a process for preparing exo-maltotetraohydrolase of Pseudomonas stutzeri or P. saccharophila using genetic engineering techniques.
U.S. Pat. No. 5,204,254 describes a native and a genetically modified exo-maltopentao-hydrolase of an alkalophilic bacterium (DSM 5853).
Very few product-specific amylases active at high pH have been identified. Examples of those that have been identified include amylases from Bacillus sp. H-167 producing maltohexaose [22, 23], from a bacterial isolate (163-26, DSM 5853) producing maltopentaose [24], from Bacillus sp. IMD370 producing maltotetraose and smaller malto-oligosaccharides [25], and from Bacillus sp. GM 8901 that initially produced maltohexaose from starch which was converted to maltotetraose during extended hydrolysis periods [26].
Starch granules heated in the presence of water undergo an order-disorder phase transition called gelatinization, where liquid is taken up by the swelling granules. Gelatinization temperatures vary for different starches and depend for the native, unmodified starches on their biological source.
Cooling converts the gelatinised phase into a viscoelastic paste or elastic gel, depending on the starch concentration. During this process, amylose and amylopectin chains reassociate to form a more ordered structure. With time, more associations are formed and they become even more ordered. It is believed that associations of amylopectin chains DP 15-20 lead to a thermoreversible, quasi-crystalline structure.
In consequence of detrimental retrogradation, the water-holding capacity of the paste or gel system is changed with important implications on the gel texture and dietary properties.
It is known that the quality of baked bread products gradually deteriorates during storage. The crumb loses softness and elasticity and becomes firm and crumbly. This so-called staling is primarily due to the detrimental retrogradation of starch, which is understood to be a transition of the starch gelatinised during baking from an amorphous state to a quasi crystalline state. The increase in crumb firmness is often used as a measure of the staling process of bread.
Upon cooling of freshly baked bread the amylose fraction, within hours, retrogrades to develop a network. This process is beneficial in that it creates a desirable crumb structure with a low degree of firmness and improved slicing properties. More gradually crystallisation of amylopectin takes place within the gelatinised starch granules during the days after baking. In this process amylopectin is believed to reinforce the amylose network in which the starch granules are embedded. This reinforcement leads to increased firmness of the bead crumb. This reinforcement is one of the main causes of bread staling.
The rate of detrimental retrogradation or crystallisation of amylopectin depends on the length of the side chains of amylopectin. In accordance with this, cereal amylopectin retrogrades at a slower rate than amylopectin from pea or potato, which has a longer average chain length than cereal amylopectin.
This is supported by observations from amylopectin gel systems that amylopectin with average chain length of DP, i.e. degree of polymerisation, ≦11 do not crystallise at all. Furthermore the presence of very short chains of DP 6-9 seems to inhibit the crystallisation of surrounding longer side chains probably because of steric hindrance. Thereby these short chains seem to have a strong anti-detrimental retrogradation effect. In accordance with this, amylopectin retrogradation is directly proportional to the mole fraction of side chains with DP 14-24 and inversely proportional to the mole fraction of side chains with DP 6-9.
In wheat and other cereals the external side chains in amylopectin are in the range of DP 12-19. Thus, enzymatic hydrolysis of the amylopectin side chains can markedly reduce their crystallisation tendencies.
It is known in the art to retard the staling of bread by using glucogenic and maltogenic exo-amylases—such as amylogycosidases which hydrolyse starch by releasing glucose—and maltogenic exoamylases or β-amylases—which hydrolyse starch by releasing maltose from the non-reducing chain ends.
In this respect, Jakubczyk et al. (Zesz. Nauk. Sck. GI.Gospod Wiejsk. Warzawie, Technol. Reino-Spozyw, 1973, 223-235) reported that amyloglucosidase can retard staling of bread baked on wheat flour.
JP-62-79745 and JP-62-79746 state that the use of a β-amylase produced by Bacillus stearothermophilus and Bacillus megaterium, respectively may be effective in retarding staling of starchy foods, including bread.
EP-A412,607 discloses a process for the production of a bread product having retarded staling properties by the addition to the dough of a thermostable exoamylase, which is not inactivated before gelatinization. Only amyloglycosidases and β-amylases are listed as suitable exoamylases to be used. The exoamylase is in an amount which is able to modify selectively the crystallisation properties of the amylopectin component during baking by splitting off glucose or maltose from the non-reducing ends of amylose and amylopectin. According to EP-A-412,607, the exoamylase selectively reduces the crystallisation properties of amylopectin, without substantially effecting the crystallisation properties of amylose.
EP-A-494,233 discloses the use of a maltogenic exoamylase to release maltose in the α-configuration and which is not inactivated before gelatinization in a process for the production of a baked product having retarded staling properties. Only a maltogenic α-amylase from Bacillus strain NCIB 11837 is specifically disclosed. Apparently, the maltogenic exoamylase hydrolyses (1→4)-α-glucosidic linkages in starch (and related polysaccharides) by removing a-maltose units from the non-reducing ends of the polysaccharide chains in a stepwise manner.
Thus, the prior art teaches that certain glucogenic exoamylases and maltogenic exoamylases can provide an antistaling effect by selectively reducing the detrimental retrogradation tendencies of amylopectin through shortening of the amylopectin side chains.
Nevertheless, there is still a need to provide different and effective, preferably more effective, means for retarding the detrimental retrogradation, such as retarding the staling, of starch products, in particular baked products, more in particular bread products.